A number of proteins having biological activity, e.g. blood coagulation factors such as Factor VII, Factor VIII, Factor IX, Factor X, and Factor XIII, immunoglobulins or serum albumin have traditionally been isolated from body fluids such as serum as their only source. However, as some of the proteins are only present in small amounts, a vast number of donors have to be involved in order to allow isolation of the proteins in industrial scale. This has given rise to increased concern due to the risk of transferring blood borne diseases which together with the limitation on the natural resources has enhanced the search for alternative methods for producing such proteins in industrial scale without such risks.
Hemophilia A is an X-chromosome-linked inherited disease which afflicts 1-2 males per 10,000. The disease is caused by an absence of deficiency of Factor VIII:C. Factor VIII:C is a very large glycoprotein (native M.sub.r 330K-360K), which is present in plasma at extremely low concentrations. It is a necessary element in the proteolytic cascade which converts soluble fibrinogen to insoluble fibrin, forming a clot to prevent blood loss from traumatized tissue. In the bloodstream, it is found in noncovalent association with von Willebrand factor (vWF) which acts as a stabilizing carrier protein. Factor VIII:C is very susceptible to cleavage by thrombin, plasmin, activated protein C, and other serine proteases. It is generally isolated from plasma or plasma products as a series of related polypeptides ranging from M.sub.r 160K-40K with predominant species of M.sub.r 92K and M.sub.r 80K-77K. This complex pattern has made the analysis of the structure of active Factor VIII:C very difficult.
Recombinant proteins produced according to the invention may be Factor VII, Factor VIII, Factor IX, Factor X, and Factor XIII which may be used for substitution therapy in individuals having deficiencies in the coagulation cascade, immunoglobulin which may be used for treating infectious diseases or serum albumin which may be used as substitute for blood transfusion during operations or a constituent of a culture medium.
Recombinant proteins having Factor VIII:C activity being prepared according to the present invention may be full length Factor VIII:C corresponding to the protein isolated from plasma, or a derivative thereof having the capability of normalizing the insufficient blood clotting caused by deficiency of Factor VIII:C. The derivatives of Factor VIII:C may be shortened single chain forms or derivatives comprising two chains. Even fragments of Factor VIII:C which may not per se show coagulant activity, but which may be used in the treatment of hemophiliacs e.g. for saturation of antibodies against Factor VIII:C present in inhibitor patients.
The proteins produced in accordance with the present invention show homology with all or a part of the natural Factor VIII:C molecule.
The preparation of recombinant proteins having Factor VIII:C activity by recombinant techniques has inter alia been disclosed in a number of patent publications. Thus, European Patent Application No. 160 457 and International Patent Application No. WO 86/01961 disclose the production of full length Factor VIII:C, and European Patent Application No. EP 150 735, International Patent Application No. WO 86/06101, European Patent Application No. EP 232 112, International Patent Application No. WO 87/04187, International Patent Application No. WO 87/07144, International Patent Application No. WO 88/00381, European Patent Application No. EP 251 843, European Patent Application No. EP 253 455, European Patent Application No. EP 254 076, U.S. Pat. No. 4,980,456, European Patent Application No. EP 294 910, European Patent Application No. EP 265 778, European Patent Application No. EP 303 540, International Patent Application No. WO 91/07490, and International Patent Application No. WO 91/09122 disclose recombinant expression of subunits of Factor VIII:C or co-expression of subunits for the production of complexes showing coagulant activity or binding affinity to antibodies inhibiting Factor VIII:C.
Expression of recombinant full length human Factor VIII:C is usually low and the molecule is unstable due to proteolysis.
Derivatives of Factor VIII:C in the form of shortened single chain forms or derivatives comprising two chains have also been successfully produced by recombinant techniques. Although these derivatives normally are expressed in a higher yield than full-length Factor VIII:C, there is still a desire for increasing the level of expression.
In order to obtain an acceptable level of expression and an acceptable stability of a produced recombinant protein having biological activity it is often preferred to express the proteins in mammalian cells. These cells are usually grown in media containing mammalian serum e.g. new borne or fetal bovine serum, in amounts of about 10% serum by volume relative to total media volume. The recombinant protein is secreted into the culture medium and has to be separated from the serum components during the isolation.
However, serum has high contents of proteins which renders the recovery and purification of the desired proteins troublesome. Such addition may also introduce a potential risk of introducing malign viruses from the bovine serum into the final blood preparations. Hence, there is a need to find serum-free culture media or media having reduced amounts of serum for culturing the host cells without essentially reducing the expression or the stability of the desired protein.
Several methods of expressing proteins having biological activity in serum-free media has already been proposed.
WO 87/04187 and EP 251 843 disclose that expression of Factor VIII:C in serum-free medium in the presence of von Willebrand Factor (vWF) or phospholipid increases the expression of Factor VIII:C. In EP 254076 it is disclosed that addition of lipoprotein to a serum-free medium increases the expression of Factor VIII in a host cell carrying the gene encoding Factor VIII. JP 61 063283 discloses that expression of i.a. monoclonal antibodies in serum-free media may be increased by addition of lipoprotein in the form of an egg yolk fraction. Finally, EP 441 695 discloses the expression of Factor VIII:C or an analogue thereof in serum-free medium in the presence of a cationic or anionic polymer, preferably a polysaccharide which most preferred is in a sulphatized form. It has been proposed to accelerate the growth of Myobacterium cultures or to stabilize media for cultivating tuberculosis bacilli by adding whole egg yolk.
The present invention is based on the surprising finding that a fraction of egg yolk not being freed from lipids may be used for increasing the expression of recombinant proteins having biological activity, preferably Factor VIII:C, in serum-free media.